Method for measurement using sodium azide

ABSTRACT

A method of measuring an analyte in a sample with excellent sensitivity using a redox reaction is provided. In this method, a reducing substance or an oxidizing substance derived from the analyte is measured in the presence of a tetrazolium compound and sodium azide using the redox reaction, and an amount of the analyte is determined from the amount of the reducing substance or oxidizing substance thus measured. The tetrazolium compound and the sodium azide are present at a ratio in a range from 20:3 to 20:12. Preferably, a solution containing the tetrazolium compound and the sodium azide is aged and then added to the sample.

TECHNICAL FIELD

The present invention relates to a method for measurement using a redoxreaction.

BACKGROUND ART

Conventionally, measurement of the amount of an analyte in a sampleusing a redox reaction has been utilized for a wide range ofapplications. For example, such measurement has been utilized formeasuring glycated proteins in applications such as biochemicalanalyses, clinical tests, and the like.

For example, glycated proteins in blood, especially glycated hemoglobinsin erythrocytes, serve as important indexes in the diagnosis, treatment,etc. of diabetes, because they reflect the patient's past history ofblood glucose levels. Such glycated proteins in erythrocytes aremeasured utilizing a redox reaction, for example, in the followingmanner.

First, erythrocytes are hemolyzed to prepare a sample. Then, thishemolyzed sample is treated with a fructosyl amino acid oxidase(hereinafter referred to as “FAOD”) so that the FAOD acts on a glycationsite of a glycated protein to form hydrogen peroxide. The amount of thehydrogen peroxide corresponds to the amount of the glycated protein.Subsequently, a peroxidase (hereinafter referred to as “POD”) and areducing agent are added to the sample, so that a redox reaction occursbetween the hydrogen peroxide and the reducing agent with the POD as acatalyst. At this time, when a reducing agent that develops color whenit is oxidized is used, the amount of the hydrogen peroxide can bedetermined by measuring the color developed. As a result, the amount ofthe glycated protein in the erythrocytes can be determined.

DISCLOSURE OF INVENTION

However, depending on the sample used, the conventional methods may notexhibit sufficient measurement sensitivity and thus may fail to improvethe accuracy of the measurement. Furthermore, since glycated proteins inblood serve as important indexes in the diagnosis, treatment, etc. ofdiabetes as described above, still further improvement in the accuracyof measurement is desired in methods of measuring them using a redoxreaction.

Therefore, it is an object of the present invention to provide a methodof measuring an analyte in a sample with high sensitivity using a redoxreaction.

In order to achieve the above object, the present invention provides amethod of measuring an analyte in a sample using a redox reaction,including: measuring an amount of a reducing substance or an oxidizingsubstance derived from the analyte in the presence of a tetrazoliumcompound and sodium azide using the redox reaction; and determining anamount of the analyte from the amount of the reducing substance oroxidizing substance thus measured. By carrying out the measurement inthe presence of the tetrazolium compound and the sodium azide asdescribed above, the measurement sensitivity can be improved, althoughthe mechanism is unknown. In the present invention, “a reducingsubstance or an oxidizing substance derived from an analyte” includesthe analyte itself, a reducing/oxidizing substance contained therein,and a reducing/oxidizing substance formed from the analyte using anoxidoreductase or the like.

In the method of the present invention, it is preferable that thetetrazolium compound (A) and the sodium azide (B) are present at a ratio(molar ratio A:B) in a range from 20:3 to 20:12.

In the method of the present invention, it is preferable that a finalconcentration of the tetrazolium compound in a reaction solution of theredox reaction is in a range from 0.5 to 2.5 mmol/l, and a finalconcentration of the sodium azide in the reaction solution is in a rangefrom 0.13 to 1.3 mmol/l.

In the method of the present invention, it is preferable that a solutioncontaining the tetrazolium compound and the sodium azide is aged and isthen added to the sample because this allows still further improvementin the sensitivity.

It is preferable that the solution is aged at a temperature in a rangefrom 20° C. to 60° C. Furthermore, it is preferable that the solution isaged for at least 6 hours, more preferably for 6 to 120 hours.

In the method of the present invention, it is preferable that thetetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt.

In the method of the present invention, it is preferable that theoxidizing substance derived from the analyte is hydrogen peroxide, andthat the amount of the hydrogen peroxide is measured by the redoxreaction. The amount of the hydrogen peroxide preferably is measuredusing an oxidase and a substrate that develops color by oxidation(hereinafter, referred to as a color-developing substrate), for example.

In the method of the present invention, the type of the sample is notparticularly limited. The method also can be applied to samples otherthan whole blood, plasma, serum, and blood cells, e.g., biologicalsamples such as urine and spinal fluid, drinks such as juices, foodssuch as soy sauce and Worcestershire sauce.

Furthermore, the analyte is not particularly limited as long as a redoxreaction is utilized. For example, the analyte may be components inwhole blood, components in erythrocytes, components in plasma,components in serum, components in urine, components in spinal fluid,and the like, and it is preferably a component in erythrocytes. Forexample, when a component in erythrocytes is to be measured, whole blooditself may be hemolyzed to prepare a sample, or erythrocytes may beseparated from whole blood and hemolyzed to prepare a sample. Examplesof the analyte include glycated proteins such as glycated hemoglobinsand glycated albumins, glycated peptides, glycated amino acids, glucose,uric acid, cholesterol, creatinine, sarcosine, and glycerol. Amongthese, glycated proteins are more preferable.

In the method of the present invention, when the analyte is a glycatedprotein, it is preferable that a glycation site thereof is degraded byoxidation with FAOD so that hydrogen peroxide is formed. Also, when theanalyte is a glycated peptide or a glycated amino acid, it is preferablethat the glycated peptide or the glycated amino acid similarly issubjected to the action of FAOD. Moreover, it is preferable thatglycated proteins and glycated peptides are treated with a proteaseprior to the FAOD treatment as necessary.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the correlation between a molar ratio at whicha tetrazolium compound and sodium azide are added, a Hb concentration,and an absorbance in one example of a method for measurement accordingthe present invention.

FIG. 2 is a graph showing the relationship between an aging period andan absorbance of a solution containing a tetrazolium compound and sodiumazide in another example of a method for measurement according thepresent invention.

FIG. 3 is a graph showing the relationship between an aging period andan absorbance of a solution containing a tetrazolium compound and sodiumazide in still another example of a method for measurement according thepresent invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The tetrazolium compound used in the present invention preferablycontains ring substituents at least at two positions on its tetrazolering, more preferably at three positions on its tetrazole ring, forexample.

In the case where the tetrazolium compound contains ring substituents atleast at two positions on its tetrazole ring as described above, it ispreferable that the ring substituents are at the 2-position and3-position on the tetrazole ring. Further, in the case where thetetrazolium compound contains ring substituents at three positions onits tetrazole ring, it is preferable that the ring substituents are atthe 2-position, 3-position, and 5-position on the tetrazole ring.

Further, it is preferable that at least two ring substituents of thetetrazolium compound have a benzene ring structure. Other than thebenzene ring structure, the ring substituents may have a resonancestructure with S or O being contained in the ring skeleton, for example.Examples of the ring substituents with such a resonance structureinclude a thienyl group, thiazoyl group, and the like.

Furthermore, it is preferable that the tetrazolium compound containsring substituents at least at three positions on its tetrazole ring andat least two of the ring substituents have a benzene ring structure.

Still further, it is preferable that at least one ring substituentcontains a functional group, and a larger number of functional groupsare more preferable.

As the functional group, an electron-withdrawing functional grouppreferably is used. For example, a halogen group, ether group, estergroup, carboxy group, acyl group, nitroso group, nitro group, hydroxygroup, sulfo group, and the like can be used. Other than these,characteristic groups containing oxygen such as a hydroperoxy group, oxygroup, epoxy group, epidioxy group, oxo group, and the like; andcharacteristic groups containing sulfur such as a mercapto group,alkylthio group, methylthiomethyl group, thioxo group, sulfino group,benzenesulfonyl group, phenylsulfonyl group, p-toluenesulfonyl group,p-tolylsulfonyl group, tosyl group, sulfamoyl group, isothiocyanategroup, and the like also can be used, for example. Among theseelectron-withdrawing functional groups, a nitro group, sulfo group,halogen group, carboxy group, hydroxy group, methoxy group, ethoxy groupare preferable. Further, in addition to the above-describedelectron-withdrawing functional groups, unsaturated hydrocarbon groupssuch as a phenyl group (C₆H₅—), styryl group (C₆H₅CH═CH—), and the likealso can be used, for example. It is to be noted that the functionalgroups may have been ionized by dissociation.

Still further, it is preferable that the tetrazolium compound containsbenzene rings at the 2-position and 3-position on its tetrazole ring andat least one of the benzene rings contains at least one functional groupselected from the group consisting of a halogen group, carboxy group,nitro group, hydroxy group, sulfo group, methoxy group, and ethoxygroup. It is to be noted here that both the benzene rings may have sucha functional group. Further, the functional group may be contained atany positions (ortho-, meta-, pra-) on each of the benzene rings.Furthermore, the number of the functional groups is not particularlylimited, and the benzene ring may have either the same or differentfunctional groups.

Examples of the tetrazolium compound containing ring substituents havinga benzene ring structure at the 2-position, 3-position, and 5-positionon its tetrazole ring include:

-   2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium    salt;-   2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium    salt;-   2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium    salt;-   2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium salt;-   3,3′-(1,1′-biphenyl-4,4′-diyl)-bis(2,5-diphenyl)-2H-tetrazolium    salt;-   3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium    salt];-   2,3-diphenyl-5-(4-chlorophenyl) tetrazolium salt;-   2,5-diphenyl-3-(p-diphenyl) tetrazolium salt;-   2,3-diphenyl-5-(p-diphenyl) tetrazolium salt;-   2,5-diphenyl-3-(4-styrylphenyl) tetrazolium salt;-   2,5-diphenyl-3-(m-tolyl) tetrazolium salt; and-   2,5-diphenyl-3-(p-tolyl) tetrazolium salt.

The tetrazolium compound is not limited to those described above. Inaddition to the above-described tetrazolium compounds, a tetrazoliumcompound containing ring substituents having a benzene ring structure attwo positions and a ring substituent having a structure other than thebenzene ring structure at one position on its tetrazole ring also may beused. Examples of such a tetrazolium compound include:

-   2,3-diphenyl-5-(2-thienyl) tetrazolium salt;-   2-benzothiazoyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethyl    carbamoyl) phenyl]-2H-tetrazolium salt;-   2,2′-dibenzothiazoyl-5,5′-bis    [4-di(2-sulfoethyl)carbamoylphenyl]-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazolium    salt; and-   3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium salt.

Further, a tetrazolium compound containing ring substituents having abenzene ring structure at two positions and a substituent not having aring structure at one position on its tetrazole ring also can be used.Examples of such a tetrazolium compound include:

-   2,3-diphenyl-5-cyano tetrazolium salt;-   2,3-diphenyl-5-carboxy tetrazolium salt;-   2,3-diphenyl-5-methyltetrazolium salt; and-   2,3-diphenyl-5-ethyl tetrazolium salt.

Among the above-described tetrazolium compounds, the tetrazoliumcompounds containing three ring substituents are preferable as describedabove. Among these, the tetrazolium compounds containing three ringsubstituents having a benzene ring structure and a large number ofelectron-withdrawing functional groups is more preferable, and2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt is most preferable. It is to be noted here that the above-describedtetrazolium compounds may be a salt or may have been ionized, forexample.

As the FAOD, FAOD catalyzing a reaction represented by Formula (1) belowpreferably is used.R¹—CO—CH₂—NH—R²+H₂O+O₂→R¹—CO—CHO+NH₂—R²+H₂O₂  (1)

In Formula (1), R¹ denotes a hydroxyl group or a residue derived fromthe sugar before glycation (i.e., sugar residue). The sugar residue (R¹)is an aldose residue when the sugar before glycation is aldose, and is aketose residue when the sugar before glycation is ketose. For example,when the sugar before glycation is glucose, it takes a fructosestructure after glycation by an Amadori rearrangement. In this case, thesugar residue (R¹) becomes a glucose residue (an aldose residue). Thissugar residue (R¹) can be represented, for example, by—[CH(OH)]_(n)—CH₂OHwhere n is an integer of 0 to 6.

In Formula (1), R² is not particularly limited. However, when thesubstrate is a glycated amino acid, a glycated peptide, or a glycatedprotein, for example, there is a difference between the case where anα-amino group is glycated and the case where an amino group other thanthe α-amino group is glycated.

In Formula (1), when an α-amino group is glycated, R² is an amino acidresidue or a peptide residue represented by Formula (2) below.—CHR³—CO—R⁴   (2)

In Formula (2), R³ denotes an amino-acid side chain group. R⁴ denotes ahydroxyl group, an amino acid residue, or a peptide residue, and can berepresented, for example, by Formula (3) below. In Formula (3), n is aninteger of 0 or more, and R³ denotes an amino-acid side chain group asin the above.—(NH—CHR³—CO)_(n)—OH  (3)

In Formula (1), when an amino group other than the α-amino group isglycated (i.e., an amino-acid side chain group is glycated), R² can berepresented by Formula (4) below.—R⁵—CH(NH—R⁶)—CO—R⁷  (4)

In Formula (4), R⁵ denotes a portion other than the glycated amino groupin the amino-acid side chain group. For example, when the glycated aminoacid is lysine, R⁵ is as follows.—CH₂—CH₂—CH₂—CH₂—For another example, when the glycated amino acid is arginine, R⁵ is asfollows.—CH₂—CH₂—CH₂—NH—CH(NH₂)—

In Formula (4), R⁶ denotes hydrogen, an amino acid residue, or a peptideresidue, and can be represented, for example, by Formula (5) below. InFormula (5), n denotes an integer of 0 or more, and R³ denotes anamino-acid side chain group as in the above.—(CO—CHR³—NH)_(n)—H  (5)

In Formula (4), R⁷ denotes a hydroxyl group, an amino acid residue, or apeptide residue, and can be represented, for example, by Formula (6)below. In Formula (6), n is an integer of 0 or more, and R³ denotes anamino-acid side chain group as in the above.—(NH—CHR³—CO)_(n)—OH  (6)

Examples of the FAOD include those produced by the following genera, forexample: the genus fusarium, the genus Gibberella, the genusPenicillium, the genus Armillaria, the genus Caldariomyces, the genusGanoderma, and the genus Aspergillus. Specific examples include Fusariumoxysporum S-1F4 (FERM BP-5010), Fusarium oxysporum f. sp. lini(IFO NO.5880), Fusarium oxysporum f. sp. batatas (IFO NO. 4468), Fusariumoxysporum f. sp. niveum (IFO NO. 4471), Fusarium oxysporum f. sp.cucumerium (IFO NO. 6384), Fusarium oxysporum f. sp. melongenae (IFO NO.7706), Fusarium oxysporum f. sp. apii (IFO NO. 9964), Fusarium oxysporumf. sp. pini (IFO NO. 9971), Fusarium oxysporum f. sp. fragariae (IFO NO.31180), Gibberella fujikuroi (IFO NO. 6356, 6605), Penicilliumjanthinellum S-3413 (FERM BP-5475), Penicillium janthinellum (IFO NO.4651, 6581, 7905), Penicillium oxalicum (IFO NO. 5748), Penicilliumjavanicum (IFO NO. 4639), Penicillium chrysogenum (IFO NO. 4897),Penicillum cyaneum (IFO NO. 5337), Aspergillus terreus (IFO NO. 6365),Aspergillus terreus GP- 1 (FERM BP-5684), Aspergillus oryzae (IFO NO.4242), and Aspergillus oryzae (IFO NO. 5710).

Furthermore, examples of commercially available FAOD include a productnamed Fructosyl-Amino Acid Oxidase (FAOX-E) (Kikkoman Corporation) and aproduct named Fructosyl Amine Oxidase (Asahi Chemical Industry Co.,Ltd.), which specifically act on a glycated amino acid having a glycatedα-amino group.

Hereinafter, the method of the present invention will be described indetail with reference to the following examples, in which a glycatedprotein in blood cells is measured.

First, whole blood itself is hemolyzed, or a blood cell fraction isseparated from whole blood in the usual way such as centrifugation andthen hemolyzed, so as to prepare a hemolyzed sample. The method ofcausing the hemolysis is not particularly limited, and can be, forexample, a method using a surfactant, a method using ultrasonic waves, amethod utilizing a difference in osmotic pressure, and a method using afreeze-thawing technique. Among these, the method using a surfactant ispreferable because of its simplicity in operation, etc.

As the surfactant, for example, non-ionic surfactants such aspolyoxyethylene-p-t-octylphenyl ether (e.g. Triton series surfactants),polyoxyethylene sorbitan alkyl ester (e.g. Tween series surfactants),polyoxyethylene alkyl ether (e.g. Brij series surfactants), and the likecan be used. Specific examples are Triton X-100, Tween-20, Brij 35, andthe like. The conditions of the treatment with the surfactant usuallyare as follows: when the concentration of blood cells in the solution tobe treated is in the range from 1 to 10 vol %, the surfactant is addedso that its concentration in the solution falls in the range from 0.01to 5 wt %, and stirred at room temperature for about several seconds(about 5 seconds) to 10 minutes.

Next, a tetrazolium compound and sodium azide are added to the hemolyzedsample.

By adding the tetrazolium compound and sodium azide, the sensitivitybecomes about 1.2 to 3 times greater than in the case where they are notadded.

When the concentration of blood cells in the solution to be treated isin the range from 0.2 to 2 vol %, the tetrazolium compound preferably isadded so that its concentration in the solution falls in the range from0.005 to 400 mmol/l, more preferably from 0.02 to 100 mmol/l, andparticularly preferably from 0.1 to 50 mmol/l. Specifically, when thetetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt, it preferably is added so that its concentration falls in therange from 0.004 to 16 mmol/l, more preferably from 0.02 to 10 mmol/l,and particularly preferably from 0.1 to 5 mmol/l. Moreover, thetetrazolium compound may be used either alone or in combinations of twoor more types.

Furthermore, the tetrazolium compound (A) and the sodium azide (B) areadded so that they are present at a ratio (molar ratio A: B), forexample, in the range from 20:3 to 20:12, preferably 20:5 to 20:11, andmore preferably 20:6 to 20:10.

The tetrazolium compound and sodium azide may be added to the hemolyzedsample simply as they are. However, in terms of simplicity in operationetc., it is preferable to use a tetrazolium compound solution obtainedby dissolving the tetrazolium compound in a solvent and a sodium azidesolution obtained by dissolving the sodium azide in a solvent, or aliquid mixture containing both the tetrazolium compound and sodium azide(i.e., a tetrazolium compound-sodium azide liquid mixture).

The concentration of the tetrazolium compound (C) or the sodium azide(D) in the above-described respective solutions can be determined asappropriate depending on the diluting factor of the solutions when theyare added to the hemolyzed sample, etc., but the concentration of thetetrazolium compound (C) is, for example, in the range from 0.6 to 10mmol/l, preferably from 0.75 to 3 mmol/l, and more preferably from 1 to2.4 mmol/l. When the liquid mixture is to be used, the liquid mixturecontains the tetrazolium compound (C) and the sodium azide (D), forexample, at a ratio (molar ratio C:D) in a range from 20:5 to 20:11,preferably 20:6 to 20:8.

As the solvent of the above-described solutions, Good's buffers such asMOPS, MES, MOPSO, DIPSO, TES, POPSO, and HEPES, a phosphate buffer, andthe like can be used, for example. Among these, MES and MOPS arepreferable. The pH of the solvent is, for example, in the range from 5.0to 7.0, preferably 5.5 to 6.5, and more preferably 5.5. Theconcentration of the buffer is, for example, in the range from 1 to 100mmol/l, preferably 1 to 10 mmol/l. The final concentration of the bufferafter being added to the hemolyzed sample is, for example, in the rangefrom 0.7 to 9 mmol/l, preferably from 0.8 to 4.5 mmol/l.

Moreover, the tetrazolium compound-sodium azide liquid mixture preparedpreferably is left for a certain period before being added to thehemolyzed sample so as to be aged because this allows still furtherimprovement in sensitivity. According to this aging treatment, thesensitivity becomes, for example, about 1.2 to 3 times greater than inthe case where the aging treatment is not performed.

In the aging treatment, the treatment temperature preferably is in therange from 40° C. to 60° C., more preferably 50° C. to 60°, and thetreatment period is, for example, at least 6 hours, preferably 6 to 120hours, and more preferably 6 to 72 hours.

After the tetrazolium compound and sodium azide are added to thehemolyzed sample simply as they are or as the above-described solution,the pretreatment of the hemolyzed sample is carried out, usually byincubating the sample at 40° C. to 60° C. for 6 to 72 hours. Bypretreating the sample with the tetrazolium compound, the influence ofreducing substances and the like contained in the sample on a redoxreaction can be eliminated, whereby the accuracy of measurement isimproved. Although the tetrazolium compound contributes to theimprovement in the accuracy of measurement as described above, it isnecessary that sodium azide coexists with the tetrazolium compound inorder to achieve the improvement in measurement sensitivity as an objectof the present invention. By using the tetrazolium compound and sodiumazide in combination, an effect peculiar to the present invention can beobtained.

Next, the pretreated hemolyzed sample containing the tetrazoliumcompound and sodium azide is treated with a protease. This proteasetreatment is carried out so that FAOD to be used later can act on theanalyte more easily.

The type of the protease is not particularly limited, and for example,serine proteases, thiol proteases, metalloproteinases, and the like canbe used. Specifically, trypsin, proteinase K, chymotrypsin, papain,bromelain, subtilisin, elastase, aminopeptidase, and the like arepreferable. In the case where the glycated protein to be degraded is aglycated hemoglobin, the protease is the one that degrades the glycatedhemoglobin selectively, and bromelain, papain, trypsin derived fromporcine pancreas, metalloproteinases, and protease derived from Bacillussubtilis, and the like are preferable. Examples of the protease derivedfrom Bacillus subtilis include a product named Protease N (e.g., FlukaChemie AG), a product named Protease N “AMANO” (Amano Enzyme Inc.), andthe like. Examples of the metalloproteinases include metalloproteinase(EC 3. 4. 24. 4) derived from the genus Bacillus (e.g., a product namedToyoteam manufactured by Toyobo Co., Ltd.) and the like. Among these,metalloproteinases, bromelain, and papain are more preferable, andmetalloproteinases are particularly preferable. Thus, a degradationproduct of a specific protein can be prepared selectively by using aprotease that degrades the protein selectively. The protease treatmentusually is carried out in a buffer, and the conditions of the treatmentare determined as appropriate depending on the type of the proteaseused, the type and the concentration of the glycated protein as ananalyte, etc.

As the buffer, CHES, CAPSO, CAPS, phosphate, Tris, EPPS, HEPES buffers,and the like can be used, for example. The pH of the buffer is, forexample, in the range from 6 to 13, preferably from 7 to 11. Moreover,the final concentration of the buffer in the solution subjected to theprotease treatment is, for example, in the range from 1.0 to 10 mmol/l.

Specifically, when the pretreated hemolyzed sample is treated using ametalloproteinase as the protease, the protease treatment usually iscarried out under the conditions as follows: the concentration of themetalloproteinase in the reaction solution in the range from 0.1 to 40MU/l; the concentration of blood cells in the reaction solution in therange from 0.05 to 15 vol %; the reaction temperature in the range from15° C. to 37° C.; the reaction period in the range from 1 minute to 24hours; and the pH in the range from 6 to 12.

Furthermore, when the pretreated hemolyzed sample is treated usingprotease K as the protease, the protease treatment usually is carriedout under the conditions as follows: the concentration of the proteasein the reaction solution in the range from 10 to 300 KU/l; theconcentration of blood cells in the reaction solution in the range from0.05 to 15 vol %; the reaction temperature in the range from 15° C. to37° C.; the reaction period in the range from 1 minute to 24 hours; andthe pH in the range from 6 to 12. Moreover, the type of the buffer isnot particularly limited, and for example, Tris-HCl buffer, EPPS buffer,PIPES buffer, and the like can be used.

Next, the degradation product obtained by the protease treatment istreated with the FAOD. The reaction shown by Formula (1) above iscatalyzed by this FAOD treatment.

Similarly to the above-described protease treatment, this FAOD treatmentpreferably is carried out in a buffer. The conditions of the FAODtreatment are determined as appropriate depending on the type of theFAOD used, the type and the concentration of the glycated protein as ananalyte, and the like.

Specifically, the FAOD treatment is carried out, for example, under thefollowing conditions: the concentration of the FAOD in the reactionsolution in the range from 50 to 50,000 U/l, the concentration of theblood cells in the reaction solution in the range from 0.01 to 1 vol %,the reaction temperature in the range from 15° C. to 37° C., thereaction period in the range from 1 to 60 minutes, and the pH in therange from 6 to 9. Moreover, the type of the buffer is not particularlylimited, and the same buffers as in the protease treatment also can beused in the FAOD treatment.

Next, the hydrogen peroxide formed by the FAOD treatment is measured bya redox reaction using POD and the color-developing substrate.

As the color-developing substrate,N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, orthophenylenediamine (OPD), a substrate in which aTrinder's reagent and 4-aminoantipyrine are combined, and the like canbe used, for example. Examples of the Trinder's reagent include phenols,phenol derivatives, aniline derivatives, naphthols, naphtholderivatives, naphthylamine, and naphthylamine derivatives. Furthermore,in place of the aminoantipyrine, it is possible to use aminoantipyrinederivatives, vanillin diamine sulfonic acid, methylbenzothiazolinonehydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH),and the like. Among these color-developing substrates,N-(carboxymethylaminocarbonyl)-4,4′-bis (dimethylamino)diphenylaminesodium salt is particularly preferable.

The redox reaction usually is carried out in a buffer. The conditions ofthe reaction are determined as appropriated depending on theconcentration of the hydrogen peroxide formed, etc. The conditions areusually as follows: the concentration of the POD in the reactionsolution in the range from 10 to 100,000 IU/l; the concentration of thecolor-developing substrate in the range from 0.005 to 30 mmol/l; thereaction temperature in the range from 15° C. to 37° C.; the reactionperiod in the range from 0.1 to 30 minutes; and the pH in the range from5 to 9. Moreover, the type of the buffer is not particularly limited,and for example, the same buffers as in the protease treatment and theFAOD treatment can be used.

In the redox reaction, for example, when the color-developing substrateis used, the amount of the hydrogen peroxide can be determined bymeasuring the degree of the color developed (i.e. absorbance) in thereaction solution with a spectrophotometer. Then, for example, theamount of the glycated protein in the sample can be determined using theconcentration of the hydrogen peroxide and a calibration curve or thelike.

The degree of the color developed can be determined not only bymeasuring the absorbance but also by optical measurement such asmeasurement of reflectance or the like. Moreover, the amount of thehydrogen peroxide can be determined not only by the above-describedenzymatic method using the POD etc. but also by an electrical method,for example.

Thus, by adding a tetrazolium compound and sodium azide, the measurementof a glycated protein using a redox reaction can be carried out withhigh sensitivity.

The order of adding a tetrazolium compound and sodium azide is notparticularly limited. However, since aging them enhances the effect ofthe present invention, they preferably are mixed with each other inadvance.

Furthermore, as described above, the analyte is not particularly limitedas long as a redox reaction is utilized. Examples of the analyte otherthan the above-described glycated proteins include glycated peptides,glycated amino acids, glucose, cholesterol, uric acid, creatinine,sarcosine, and glycerol. When the amount of each of the above-describedexamples of the analyte is measured, measurement can be carried out, forexample, by adding a tetrazolium compound and sodium azide to ameasurement sample in the same manner as described above, then forming areducing substance or an oxidizing substance derived from the analyte inthe following manner, and measuring the amount of the reducing substanceor oxidizing substance using a redox reaction.

When the measurement is carried out by forming hydrogen peroxide, thehydrogen peroxide may be formed, for example, by action of: a glucoseoxidase on the glucose; a cholesterol oxidase on the cholesterol; auricase on the uric acid; a sarcosine oxidase on the creatinine; asarcosine oxidase on the sarcosine; or a glycerol oxidase on theglycerol; respectively. The amount of the hydrogen peroxide can bemeasured in the same manner as above. Moreover, glycated peptides andglycated amino acids can be measured, for example, in the same manner asin the measurement of the glycated proteins.

Furthermore, when the amount of the analyte is determined by forming areducing substance derived from the analyte, measuring the amount of thereducing substance by a redox reaction, and then determining the amountof the analyte from the amount of the reducing substance, themeasurement can be carried out, for example, in the following manner.

When the analyte is glucose, for example, a reducing substance such asNADH or NADPH is formed using glucose dehydrogenase in the presence ofNAD, NADP, or the like. Then, the NADH or NADPH as a reducing substancederived from the analyte is measured by a redox reaction, using, forexample, diaphorase and a substrate that develops color by reduction.Then, as described above, the amount of the analyte in the sample can bedetermined, for example, using the concentration of the reducingsubstance derived from the analyte and a calibration curve or the like.Furthermore, for example, cholesterol dehydrogenase can be used when theanalyte is cholesterol, and sarcosine dehydrogenase can be used when theanalyte is sarcosine.

As the substrate that develops color by reduction, although notparticularly limited, for example, 2,6-dichlorophenolindophenol and thelike can be used. Moreover, in order to obtain measured values with moreexcellent reliability, it is preferable to measure an absorbance beforemeasuring the reducing substance derived from the analyte, for example.

EXAMPLES Example 1 and Comparative Example 1

First, blood having a Hb concentration of 150 g/l (HbA1c 5.8%) washemolyzed by adding 0.3 ml of the following hemolysis reagent to preparea hemolyzed sample. Then, 65 μl of the following first reagentscontaining 3g/l sodium azide aqueous solution at predeterminedconcentrations (0 mmol/l, 0.30 mmol/l, 0.616 mmol/l, 0.924 mmol/l, 1.232mmol/l, and 1.54 mmol/l) were added to mixtures of 25 μl of thehemolyzed sample and 20 μl of purified water, respectively. Theresultant mixtures were incubated at 37° C. for 5 minutes. The firstreagents were aged at 50° C. for 20 hours before being added to thehemolyzed sample. Subsequently, 45 μl of the following color-developingreagent A further was added, and the resultant mixtures were incubatedat 37° C. for 3 minutes. Thereafter, with regard to the thus-obtainedreaction solutions, the absorption was measured at the main wavelengthof 751 nm and the sub-wavelength of 805 nm using a biochemical automaticanalysis apparatus (product name Bio Majesty, manufactured by JapanElectron Optics Laboratory Co. Ltd.). The results are shown in Table 1.The mixture containing no sodium azide (0 g/l) was regarded asComparative Example 1.

(Hemolysis Reagent: pH 9.0) CHES buffer 380 mmol/l Polyoxyethylenelauryl ether 24 g/l (First Reagent) 100 mmol/l MOPS (pH 6.0) 0.15 ml 13MU/l metalloproteinase 0.60 ml 10 mmol/l tetrazolium compound 0.60 ml 20mmol/l CaCl₂ 0.045 ml  3 g/l NaN₃ predetermined amount distlled waterremaining portion total amount  3.0 ml

As the metalloproteinase, metalloproteinase derived from the genusBacillus was used. As the tetrazolium compound,2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt (product name WST-3, manufactured by Dojindo Laboratories,hereinafter referred to as “WST-3”) was used.

(Color-Developing Reagent A) FAOD (product name Fructosyl Amino AcidOxidase, 26.0 KU/l ARKRAY, INC., hereinafter the same) POD (Toyobo Co.,Ltd., hereinafter the same) 77.6 KU/l Color-developing substrate(product name DA-64, 0.052 mmol/l Wako Pure Chemical Industries, Ltd.,hereinafter the same) Tris-HCl buffer (pH 6.9) 200 mmol/l

TABLE 1 Final concentration Concentration of of sodium azide tetrazoliumcompound in first reagent in first reagent (mmol/l) (mmol/l) AbsorbanceCom. Ex. 1 0 2 0.0124 Ex. 1 0.300 2 0.0476 0.616 2 0.0518 0.924 2 0.04751.232 2 0.0418 1.540 2 0.0275

As can be seen from Table 1, in Example 1, by conducting the measurementin the presence of the tetrazolium compound and sodium azide, theabsorbance higher than that in Comparative Example 1 was obtainedbecause the amount of the color developed by the color-developingsubstrate DA-64 increased. Thus, it can be said that the methodaccording to the present invention can improve the measurementsensitivity.

Example 2 and Comparative Example 2

In Example 2, WST-3 and sodium azide were added so that they werepresent at various ratios, and further improvement in measurementsensitivity by aging them was confirmed.

(Reagent Sample)

Reagent samples (A) to (E) were prepared by mixing WST-3 and sodiumazide so that they were present at the following ratios. Then, thesamples (B) to (E) were incubated at 40° C. for 20 hours. Subsequently,the samples (A) to (E) were maintained at the temperature not higherthan 4° C. and metalloproteinase was added to the samples so that itsconcentration became 2.66 MU/l.

(Ratio Between WST-3 and Sodium Azide)

Example 2

(A) (B) (C) (D) (E) WST-3 (mmol/l) 2.0 2.0 2.0 2.7 1.3 NaN₃ (mmol/l) 0.70.7 1.4 1.4 0.7 WST-3:NaN₃ (molar ratio) 3:1 3:1 1.43:1 1.9:1 1.9:1 MOPSpH 6.5 (mmol/l) 5.5 5.5 5.5 5.5 5.5 CaCl₂ (mmol/l) 5.5 5.5 5.5 5.5 5.5NaCl (mmol/l) 0.33 0.33 0.33 0.33 0.33 Aging — ∘ ∘ ∘ ∘

(Hemolyzed Solution)

Distilled water was added to blood cells. The mixture was frozen andthen thawed to hemolyze the blood cells In the hemolyzed solution thusobtained, the Hb concentration was 100 g/l and HbA1c was 4.9%.

The hemolyzed solution thus prepared was pretreated by adding thefollowing pretreatment reagent so as to have the following compositions.

Hb concentration 6.7 g/l 13.4 g/l 20.1 g/l 33.5 g/l Hemolyzed solution 50 μl 100 μl 150 μl 250 μl Pretreatment reagent 434 μl 434 μl 434 μl434 μl Distilled water 266 μl 216 μl 166 μl  66 μl total amount 750 μl

The pretreatment reagent was prepared by adding a surfactant to a bufferso that its concentration became 20 wt %. As the surfactant,polyoxyethylene lauryl ether (product name Nikkol, manufactured by NihonSurfactant Kogyo K.K.) was used. The buffer used was a mixed buffer (pH9.5) obtained by mixing a glycinamide buffer and a glycine solution sothat the final concentration of glycinamide became 200 mM and the finalconcentration of glycine became 40 mM.

(Color-Developing Reagent B) FAOD 25.9 KU/l POD 77.6 KU/lColor-developing substrate 0.0518 mmol/l Tris-HCl buffer (pH 6.9) 200mmol/l

(Measuring Procedure)

First, the pretreated hemolyzed solutions were diluted 2.4-fold withdistilled water, and 20 μL of these diluted solutions were mixed with 60μl of each of the reagent samples and 50 μl of the color-developingreagent B. The resultant mixtures were allowed to react at 37° C. for 15minutes. Thereafter, the absorption was measured at the main wavelengthof 751 nm and the sub-wavelength of 805 nm using a biochemical automaticanalysis apparatus (product name JCA-BM 8, manufactured by JapanElectron Optics Laboratory Co. Ltd.). The results are shown in Table 2below and in the graph shown in FIG. 1. The graph of FIG. 1 shows therelationship between a molar ratio between the tetrazolium compound andsodium azide, a Hb concentration, and an absorbance.

TABLE 2 Example 2 Hb concentration (A) (B) (C) (D) (E)   0 g/l 0.15420.1628 0.1469 0.1403 0.1647  6.7 g/l 0.0034 0.0101 0.0087 0.0099 0.009713.4 g/l 0.0063 0.0180 0.0146 0.0173 0.0171 20.1 g/l 0.0094 0.02590.0202 0.0249 0.0243 33.5 g/l 0.0127 0.0384 0.0278 0.0374 0.0349

As shown in Table 2 and FIG. 1, high absorbance was exhibited by addingthe tetrazolium compound and sodium azide. Moreover, further improvementin absorbance was achieved by conducting incubation (aging) ((B) to(E)). Moreover, since the absorbance increased in keeping with the Hbconcentration, it can be said that the absorbance did not increase dueto the absorption of the tetrazolium compound and sodium azidethemselves but increased because the absorbance of the color-developingsubstrate DA-64 increased by adding the tetrazolium compound and sodiumazide. Furthermore, the significant increase in absorbance was observedwhen the tetrazolium compound and sodium azide were added so that theywere present at the molar ratio of 3:1 and they were aged as well. Thus,because the amount of color developed by the color-developing substrateincreased by adding the tetrazolium compound and sodium azide and itincreased still further by conducting aging, it can be said that themeasurement sensitivity was improved.

Example 3

In Example 3, a tetrazolium compound and sodium azide were aged in asolution to examine how this affects the improvement in measurementsensitivity.

First, a liquid mixture containing 3.33 mmol/l of WST-3 and 0.0833 g/lof sodium azide was prepared and then aged by being incubated at 60° C.Then, samples were taken predetermined periods (0 hour, 1 hour, 6 hours,14 hours, 16 hours, and 18 hours) after the start of the incubation.These samples were used as reagent samples.

On the other hand, a hemolyzed solution (Hb concentration: 100 g/l,HbA1c: 4.9%) was prepared in the same manner as in Example 2. Then,predetermined amounts (2 μl, 5 μl, 10 μl, 20 μl, and 30 μl) of hemolyzedsolution were mixed with 300 μl of the following pretreatment reagent toprepare substrate Hb solutions (A to E).

(Pretreatment Reagent)

Liquid Mixture (pH 9.4) of 80 mmol/l CHES and 30 mmol/l MOPS 9 g/l ofPolyoxyethylene Lauryl Ether

First, the respective substrate Hb solutions were diluted 2-fold withdistilled water, and 20 μL of these diluted solutions were mixed with 65μl of each of the following second reagents respectively containing theabove-described reagent samples and 45 μl of the color-developingreagent C. The resultant mixtures were allowed to react at 37° C. for 15minutes. Thereafter, the absorption at the main wavelength of 751 nm andthe sub-wavelength of 805 nm was measured using the above-describedbiochemical automatic analysis apparatus. The results are shown in Table3 below and FIG. 2. FIG. 2 is a graph showing the relationship betweenan aging period and an absorbance.

(Second Reagent) 1 mol/l CaCl₂ 0.035 ml 5 mol/l NaCl 0.280 ml 30 mmol/lMES (pH 5.5)  1.40 ml 100 MU/l metalloproteinase  0.7 ml distlled water0.385 ml total amount  7.0 ml (Color-Developing Reagent C) FAOD 25.9KU/l POD 77.6 KU/l Color-developing substrate 0.0518 mmol/l Tris-HClbuffer (pH 7.0) 300 mmol/l

TABLE 3 Final concentration of Hb Aging period (A) (B) (C) (D) (E)(hour) 0.10 g/l 0.25 g/l 0.50 g/l 0.96 g/l 1.40 g/l  0 0.0003 0.00070.0015 0.0037 0.0065  1 0.0003 0.0008 0.0017 0.0042 0.0073  6 0.00080.0020 0.0039 0.0084 0.0129 14 0.0009 0.0020 0.0039 0.0084 0.0130 160.0008 0.0020 0.0039 0.0083 0.0128 18 0.0008 0.0020 0.0039 0.0083 0.0129

As shown in Table 3 and FIG. 2, it was found that further improvement inmeasurement sensitivity can be achieved by using the solution containingthe tetrazolium compound and sodium azide after aging it, regardless ofthe Hb concentration. Furthermore, the sensitivity reached its maximumwhen the aging period was 6 hours or longer.

Example 4

In Example 4, a tetrazolium compound and sodium azide were aged in asolution to examine how this affects the improvement in measurementsensitivity.

First, the following liquid mixture containing WST-3 and sodium azidewas prepared and then aged by being incubated at predeterminedtemperatures (30° C., 40° C., 50° C., and 60° C.) for predeterminedperiods (0 hour, 6 hours, 15 hours, 24 hours, 48 hours, and 72 hours) toprepare reagent samples.

(Composition of Liquid Mixture) WST-3 2.20 mmol/l Sodium azide 0.05 g/lMOPS (pH 6.5) 5.50 mmol/l CaCl₂ 5.50 mmol/l NaCl 330 mmol/l

On the other hand, substrate Hb solutions (Hb concentration: 100 g/l)were prepared in the same manner as in Example 2 and diluted 2-fold (byvolume) with purified water to prepare diluted solutions. On the otherhand, metalloproteinase solutions were prepared by mixing 9 ml of theabove-described respective reagent samples with 1 ml ofmetalloproteinase (2.7 MU/l). Then, 20 μL of the diluted solutions weremixed with 65 μl of each of the metalloproteinase solutions and 45 μl ofthe color-developing reagent A. The resultant mixtures were allowed toreact at 37° C. for 15 minutes. Thereafter, the absorption was measuredat the main wavelength of 751 nm and the sub-wavelength of 805 nm usingthe above-described biochemical automatic analysis apparatus. Theresults are shown in Table 4 below and FIG. 3. FIG. 3 is a graph showingthe relationship between an aging period and an absorbance.

TABLE 4 Aging period Aging temperature (hour) 30° C. 40° C. 50° C. 60°C.  0 0.0078 0.0078 0.0078 0.0078  6 0.0211 0.0244 0.0253 0.0277 240.0259 0.0270 0.0323 0.0320 48 0.0271 0.0286 0.0288 0.0301 72 0.03020.0311 0.0311 0.0308

As shown in Table 4 and FIG. 3, the absorbance increased rapidly whenthe aging was conducted at 60° C. Therefore, it can be said that themeasurement sensitivity can be improved with shorter period of aging asthe aging temperature becomes higher.

INDUSTRIAL APPLICABILITY

As specifically described above, the method for measurement using aredox reaction according to the present invention is excellent inmeasurement sensitivity because it uses a tetrazolium compound andsodium azide. Therefore, by applying the method of the present inventionto, for example, the measurement of HbA1c contained in erythrocytes, itbecomes possible to realize the measurement with higher accuracy than inconventional methods, which further increases the importance of HbA1c asan index in the diagnosis and the like of diabetes.

1. A method of measuring an analyte in a sample using a redox reaction,comprising: aging a solution containing a tetrazolium compound andsodium azide at a temperature of at least 20° C. and not higher than 60°C. for 6 to 120 hours; then adding the aged solution to the sample;measuring an amount of a reducing substance or an oxidizing substancederived from the analyte in the presence of the tetrazolium compound andthe sodium azide using the redox reaction; and determining an amount ofthe analyte from the amount of the reducing substance or oxidizingsubstance thus measured, wherein the tetrazolium compound (A) and thesodium azide (B) are present at a ratio (molar ratio A:B) in a rangefrom 20:3 to 20:12, and the analyte is at least one selected from thegroup consisting of glycated proteins, alycated peptides, and glycatedamino acids.
 2. The method according to claim 1, wherein a finalconcentration of the tetrazolium compound in a reaction solution of theredox reaction is in a range from 0.5 to 2.5 mmol/l, and a finalconcentration of the sodium azide in the reaction solution is in a rangefrom 0.13 to 1.3 mmol/l.
 3. The method according to claim 1, wherein thetetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt.
 4. The method according to claim 1, wherein the glycated proteinsare glycated hemoglobins.
 5. The method according to claim 1, whereinthe oxidizing substance derived from the analyte is hydrogen peroxide.6. The method according to claim 5, wherein the hydrogen peroxide isformed by a reaction between a glycation site of a glycated protein anda fructosyl amino acid oxidase.
 7. The method according to claim 5,wherein the amount of the hydrogen peroxide as the oxidizing substanceis measured using an oxidase and a substrate that develops color byoxidation.
 8. The method according to claim 7, wherein the amount of thehydrogen peroxide is measured by optical measurement of a degree ofcolor developed by the substrate that develops color by oxidation. 9.The method according to claim 8, wherein the optical measurement ismeasurement of an absorbance or a reflectance.
 10. The method accordingto claim 1, wherein the solution containing the tetrazolium compound andthe sodium azide is aged at a temperature of at least 40° C.
 11. Themethod according to claim 1, wherein the solution containing thetetrazolium compound and the sodium azide is aged at a temperature in arange from 40° C. to 60° C.